CAJ | 학술논문

目的探讨胶质瘤细胞LN229中RECK作为miR-21的调控靶点,在胶质瘤侵袭性生长中的作用.方法将反义miR-21(AS-miR-21)寡核苷酸转染至人脑胶质瘤细胞LN229中.Real-time PCR检测LN229细胞中miR-21的表达量.荧光素酶实验检测miR-21对RECK的调控关系.Transwell实验评价LN229细胞侵袭能力的变化,应用Western blot检测细胞内MMP2/9和RECK蛋白水平的变化,ELISA实验检测培养基中活性MMP2/9的表达量,动物实验评价体内条件下肿瘤侵袭性的变化.结果 Real-time PCR显示转染组中miR-21的表达量与对照组相比下调60%.荧光素酶实验证明RECK是miR-21的靶点.Transwell实验证实胶质瘤细胞侵袭能力下降,Western blot和ELISA实验证实MMP2/9表达降低,动物实验及免疫荧光反映肿瘤侵袭性生长受抑制.结论反义miR-21通过上调RECK的表达而抑制恶性胶质瘤细胞的侵袭性生长.
목적 탐토효질류세포LN229중RECK작위miR-21적조공파점,재효질류침습성생장중적작용.방법 장반의miR-21(AS-miR-21)과핵감산전염지인뇌효질류세포LN229중.Real-time PCR검측LN229세포중miR-21적표체량.형광소매실험검측miR-21대RECK적조공관계.Transwell실험평개LN229세포침습능력적변화,응용Western blot검측세포내MMP2/9화RECK단백수평적변화,ELISA실험검측배양기중활성MMP2/9적표체량,동물실험평개체내조건하종류침습성적변화.결과 Real-time PCR현시전염조중miR-21적표체량여대조조상비하조60%.형광소매실험증명RECK시miR-21적파점.Transwell실험증실효질류세포침습능력하강,Western blot화ELISA실험증실MMP2/9표체강저,동물실험급면역형광반영종류침습성생장수억제.결론 반의miR-21통과상조RECK적표체이억제악성효질류세포적침습성생장.
Objective To investigate the regulation of miR - 21 on invasion growth of human glioma cells by RECK.Method The human glioma LN229 cells were transfected with AS - miR - 21 or scrambled sequences by Lipofectamine2000.Real time PCR was conducted to detect the expression of miR-21.Luciferase experiment was performed to detect the relationship between miR-21 and RECK.The expression of RECK was evaluated by Western blot.The invasion ability was evaluated by transwell assay and subcutaneous models.Western Blot, ELISA and immunofluorescence were used to estimate the changes of MMP2/9.Results The expression of miR - 21 in LN229 cells decreased after transfection with AS-miR-21. It was proved that RECK was a direct target of miR -21 by luciferase experiment.Meanwhile, the high expression of RECK protein in AS - miR -21 group conformed its important function in this mechanism.Transwell assay demonstrated decreased invasion capability of LN229 cell lines transfected with AS- miR- 21.Western blot, ELISA, and immunofluorescence demonstrated the levels of MMP2/9 were down -regulated in AS -miR -21 group compared with control and scrambled group.Conclusions AS - miR -21 could depress the invasion of glioma cells owing to up - regulating the level of RECK which could inhibit MMP2/9 activities both in vitro and vivo.